Outrunning protein diffusion to the air-water interface in cryoEM.

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• The air-water interface is a major challenge in cryoEM specimen preparation.
• Protein diffusion to this interface can lead to denaturation and artifacts.
• Vitrification is crucial for preserving biomolecular structures in their native state.

Purpose of the Study:

• To develop a novel method for rapid specimen vitrification in cryoEM.
• To overcome the limitations imposed by the air-water interface.
• To preserve the native atomic structure of proteins during sample preparation.

Main Methods:

• Spraying picoliter droplets at high speeds (hundreds of m/s) onto a liquid ethane-coated, precooled support.
• Simultaneous droplet collapse and vitrification into the amorphous phase within microseconds.
• Utilizing a rapid freezing technique to bypass air-water interface interactions.

Main Results:

• Achieved vitrification faster than protein diffusion rates to the air-water interface.
• Preserved the atomic structure of proteins in the vitrified specimens.
• Eliminated adhesion of specimens to interfaces, confirmed by tomographic reconstructions.
• Identified specimen thickness and particle orientation as new limiting factors.

Conclusions:

• Demonstrated the physical possibility of rapid vitrification to mitigate air-water interface effects.
• This technique provides a foundation for developing new cryoEM specimen preparation instruments.
• Future work should focus on controlling specimen thickness and particle orientation for improved cryoEM data.